科学网—英文版图解(Figure legends for Y/IF multiple labeling) - 胡步根的博文

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Get the most precise number on the Subaru you want. Get a Local Price. Skip to Content. If just add these two different kinds of experimental methods together it will not work, regardless doing immunofluorescent staining 1 st or Y FISH 1 st because in order to identify donor stem cell Y as marker of origin for donor or recipient derived specific tissue type cell, after both experimental procedures the both signals need to be present on the same slide otherwise the mission is impossible. If the authors use the classic denature condition for Y FISH on tissue paraffin sections as they published, 1M sodium thiocyanate at 80C for 20 mins, then neutralized with 0.

If the authors use such denature condition on cytospins, after Y FISH probably only a few nuclei left on the slide, or nothing left on the slide.

And imagine even there is such biological event- bone marrow stem cell derives into specific tissue type cell here is from BM to type II pneumocyte happening in vivo, the frequency of such event is very low even based on the authors calculation less than 1 per How to solve this technical dilemma, it took me about 6 months to solve the technical dilemma above mentioned: interphase Y FISH combined with immunofluorescent staining while keeping the tissue and cell morphology normal.

The strategy I used is to find the common window of experimental conditions that will fit for both Y FISH and immunofluorescent staining, then hard working of error and try.

Definition of detection sensitivity needs to be defined by thousands of cells on the same slide, but not by a few cells of electronic image of very small field. Merged image: besides the Y signal, some yellow, green signals are coming from autofluorescence. After examine the whole slide under fluorescent microscope, my conclusion is: there is no way for the authors to obtain their experimental results as they claimed in their paper FASEB because the Y FISH detection sensitivity is too low.

So their results of Y FISH on bone marrow are just intentionally fabricated numbers, not scientific experimental results at all. Powered by ScienceNet. The most important information of this image should show is that after all labeling is finished Y FISH, and immunofluorescent staining , as a whole picture what really looks like Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells on the alveoli how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive.

In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image.

Compared with the image of wt male control, overall spcko has much stronger autofluorescence the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section.

Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. In this field there is obvious CD45 positive cell. Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues whether or not Y can achieve similar detection sensitivity in both tissues , and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung.

This image shows chimera characteristics of such model.

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And imagine even there is such biological event- bone marrow stem cell derives into specific tissue type cell here is from BM to type II pneumocyte happening in vivo, the frequency of such event is very low even based on the authors calculation less than 1 per How to solve this technical dilemma, it took me about 6 months to solve the technical dilemma above mentioned: interphase Y FISH combined with immunofluorescent staining while keeping the tissue and cell morphology normal.

The strategy I used is to find the common window of experimental conditions that will fit for both Y FISH and immunofluorescent staining, then hard working of error and try. Definition of detection sensitivity needs to be defined by thousands of cells on the same slide, but not by a few cells of electronic image of very small field.

Merged image: besides the Y signal, some yellow, green signals are coming from autofluorescence. After examine the whole slide under fluorescent microscope, my conclusion is: there is no way for the authors to obtain their experimental results as they claimed in their paper FASEB because the Y FISH detection sensitivity is too low.

So their results of Y FISH on bone marrow are just intentionally fabricated numbers, not scientific experimental results at all. Powered by ScienceNet. The most important information of this image should show is that after all labeling is finished Y FISH, and immunofluorescent staining , as a whole picture what really looks like Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells on the alveoli how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive.

In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b.

Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image.

Compared with the image of wt male control, overall spcko has much stronger autofluorescence the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. In this field there is obvious CD45 positive cell.

Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues whether or not Y can achieve similar detection sensitivity in both tissues , and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung.

This image shows chimera characteristics of such model. Most nuclei of CD45 negative cells are labeled as Y positive, but there is still a small percentage of such cells being labeled as Y negative.

There is no single cell being SPC positive, in terms of SPC specific staining the situation is the same as I mentioned for the image of spcko male control mouse.